This application proposes pre-clinical studies towards development of an effective killed whole-cell vaccine (WCV) to combat Vibio cholerae O139. The incidence of 0139 cholera is again increasing and the lack of an effective vaccine precludes protection of inhabitants in, and travellers to, affected regions. Trial O139 WCVs have been produced, but these have proved to be less immunogenic than their O1 counterparts. The present proposal focuses on two approaches with the potential to solve the specific problem of the poor immunogenicity of the O139 WCVs tested to date. Aim 1: To evaluate the relative significance of the O- antigen capsule and the core-linked O-antigen with respect to generation of antibodies to O139 lipopolysaccharide (LPS). This line of research will ascertain whether the different LPS structure of O139 isolates, or their synthesis of a capsule, reduces their capacity to elicit (potentially-protective) antibodies to LPS. WCVs prepared from mutant strains with modified capsule/LPS phenotypes will be compared experimentally for immunogenicity and protective efficacy, to identify a combination of capsule and LPS structures optimal for induction of antibodies to O-antigen. Aim 2: To determine whether the immunogenicity of O139 WCVs can be improved by the inclusion of toxin-coregulated pili (TCP). TCP are a critical colonisation factor of pathogenic Vibrio cholerae, and antibodies to these pili are protective experimentally. Recent evidence shows that TCP are immunogenic in man, suggesting that their incorporation into WCVs might enhance immunogenicity. This will be examined by comparing the immunogenicity and protective efficacy of related TCP-positive and TCP-negative WCVs. Note that these two approaches are not mutually exclusive and could be combined, perhaps further improving vaccine efficacy. A final aim of this proposal is to establish a new cholera model in adult mice, one which would allow direct evaluation of vaccine efficacy by making it possible to directly challenge orally mice which have been immunised with various WCVs. [unreadable] [unreadable]